EP4408493A1 - Greffe de régénération nerveuse cellularisée et ses méthodes de production - Google Patents

Greffe de régénération nerveuse cellularisée et ses méthodes de production

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Publication number
EP4408493A1
EP4408493A1 EP22877515.1A EP22877515A EP4408493A1 EP 4408493 A1 EP4408493 A1 EP 4408493A1 EP 22877515 A EP22877515 A EP 22877515A EP 4408493 A1 EP4408493 A1 EP 4408493A1
Authority
EP
European Patent Office
Prior art keywords
conduit
cellularized
nerve regeneration
biodegradable polymer
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22877515.1A
Other languages
German (de)
English (en)
Other versions
EP4408493A4 (fr
Inventor
Zhiping Pang
N. Sanjeeva MURTHY
Andrew J. BORELAND
Jasmine M. GAMBOA
Jeremy M. PERRELLE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rutgers State University of New Jersey
Original Assignee
Rutgers State University of New Jersey
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Filing date
Publication date
Application filed by Rutgers State University of New Jersey filed Critical Rutgers State University of New Jersey
Publication of EP4408493A1 publication Critical patent/EP4408493A1/fr
Publication of EP4408493A4 publication Critical patent/EP4408493A4/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
    • A61L27/3891—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types as distinct cell layers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/11—Surgical instruments, devices or methods for performing anastomosis; Buttons for anastomosis
    • A61B17/1128—Surgical instruments, devices or methods for performing anastomosis; Buttons for anastomosis of nerves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3675—Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/11—Surgical instruments, devices or methods for performing anastomosis; Buttons for anastomosis
    • A61B2017/1132—End-to-end connections
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment

Definitions

  • This disclosure is related to a cellularized nerve regeneration graft for use in repairing peripheral nerve injuries, and methods of making the same.
  • Peripheral Nerve Injuries cost the global economy over $150 billion USD annually in direct medical expenditures.
  • PNI Peripheral Nerve Injuries
  • Such injuries can arise from physical trauma, cancers, or other nervous system pathologies.
  • Associated socioeconomic costs multiply considerably in a global context, particularly in developing countries where jobs involving manual labor lead to more frequent workplace injuries.
  • nerve injuries still lack much hope for complete functional recovery, often leaving patients without sensation and/or motor function.
  • a cellularized nerve regeneration graft comprising: an electrospun biodegradable polymer conduit having an exterior surface and an interior luminal space; a plurality of fibroblasts seeded to the exterior surface of the conduit; and a system comprising a hydrogel matrix or augmented hydrogel matrix and Schwann cells, wherein the system fills the interior luminal space of the conduit.
  • the augmented hydrogel matrix may comprise a hydrogel selected from the group consisting of: RADA- 16 peptides, collagen, gelatin, alginate, hyaluronic acid, or any combination thereof, combined with a growth factor, guidance cue, structural peptide, adhesion factor, other chemical agent, or any combination thereof.
  • the electrospun biodegradable polymer conduit may comprise a plurality of layers, for example, two layers with one layer having aligned biopolymer fibers and the second layer having unaligned biopolymer fibers.
  • the electrospun biodegradable polymer may be a tyrosinederived or tyrosol-derived polymer.
  • the fibroblasts may form a continuous cell layer on the exterior surface of the conduit at a concentration of greater than about 1.0 xlO 5 cells/cm 2 .
  • the Schwann cells may be substantially evenly distributed throughout the hydrogel matrix or augmented hydrogel matrix within the interior luminal space of the conduit, for example, at a concentration of about greater than 20 million cells/mL.
  • the cellularized nerve regeneration graft may comprise a plurality of channels running in a longitudinal direction of the electrospun biodegradable polymer conduit and within the system. The plurality of channels may be hollow and reinforced with Schwann cells.
  • a method of making the cellularized nerve regeneration graft includes electrospinning a formulation of a tyrosine-derived or tyrosol -derived polymer to make the electrospun biodegradable polymer conduit; culturing fibroblasts; seeding the fibroblasts on the exterior surface of the electrospun biodegradable polymer conduit; generating human Schwann cells; embedding the human Schwann cells in the hydrogel matrix or augmented hydrogel matrix to make the system; seeding the system in the interior luminal space of the electrospun biodegradable polymer conduit; and incubating the seeded conduit to make the cellularized nerve regeneration graft.
  • Also disclosed is a method of repairing an injured peripheral nerve in a patient comprising implanting the cellularized nerve regeneration graft into the patient.
  • a method of making the cellularized nerve regeneration graft having a plurality of channels running in a longitudinal direction of the electrospun biodegradable polymer conduit is disclosed.
  • That method may include: electrospinning a formulation of tyrosinederived or tyrosol-derived polymer to make the electrospun biodegradable polymer conduit; generating human Schwann cells; embedding the human Schwann cells in the hydrogel matrix or augmented hydrogel matrix to make the system; either culturing resorbable fibers with the human Schwann cells and then loading the cultured resorbable fibers into the electrospun biodegradable polymer conduit in a longitudinal arrangement, or loading the resorbable fibers into the electrospun biodegradable polymer conduit in a longitudinal arrangement, followed by adding a suspension comprising human Schwann cells into the interior luminal space of the electrospun biodegradable polymer conduit to make cultured resorbable fibers; seeding the system into the interior luminal space of the electrospun biodegradable polymer conduit and between the cultured resorbable fibers; and incubating the seeded conduit for about 1 to about 6 weeks, wherein the resorbable fibers
  • the method may further comprise: culturing fibroblasts; and seeding the fibroblasts on the exterior surface of the electrospun biodegradable polymer conduit.
  • the resorbable fibers may be made from poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), a tyrosine-derived polymer with a high concentration of polyethylene glycol) (PEG), or any combination thereof.
  • the spacing between the cultured resorbable fibers may be about 20 pm to about 100 pm.
  • FIG. 1 A-B are electron micrograph images showing aligned and unaligned fibers in a bilayered sheet.
  • FIG. 2A-B are electron micrograph images showing aligned and unaligned fibers in a bilayered sheet.
  • FIG. 3A-E is an example of the electrospun biodegradable polymer conduit having the system of augmented hydrogel matrix and embedded Schwann cells enclosed within its inner luminal space.
  • FIG. 4A-F is an example of various 3-D printed parts used to assemble a cellularized nerve regeneration graft of the disclosure.
  • FIG. 5 is a schematic demonstrating an example of how Schwann cells may be cultured onto resorbable fibers to make longitudinal channels within a polymer conduit.
  • FIG. 6 is a schematic for making a cellularized nerve regeneration graft of the disclosure.
  • FIG. 7A-D is an example of the construction of a multi-layer biopolymer fixture for initial cell culture.
  • FIG. 8A-E is an example of the construction of a tyrosine-derived biodegradable polymer conduit and fixture assembly.
  • FIG. 9A-C is an example of a method of generating human Schwann cells, and graph of the Schwann Cell fold expression.
  • FIG. 10 depicts a second method of generating human Schwann cells using transcription factors delivered by lentivirus in combination with growth factors to drive differentiation of either iPSCs or fibroblasts (FBs) to Schwann cells.
  • FIG. 11A-C is a representation of Schwann cells and fibroblasts cultured onto opposite sides of an electrospun biodegradable polymer fiber scaffold.
  • FIG. 12 is a scheme for implantation of a cellularized nerve regeneration graft of the disclosure into a murine peripheral nerve injury model.
  • FIG. 13 is an example of magnification imaging of rat Schwann cells growing on fibers made from a tyrosine-derived polycarbonate.
  • FIG. 14A-C are cross-sectional confocal microscopy images demonstrating rat Schwann cells proliferating within collagen hydrogel matrix; tyrosine-derived polymer fibers are shown dispersed throughout the matrix, maintaining longitudinal tracts in the hydrogel matrix.
  • a method for generating a biodegradable polymer conduit seeded with neuronal support cells to facilitate axonal regeneration in PNI patients includes construction of a cellularized nerve regeneration graft (CNRG) that is suitable for connecting the injured peripheral nerve tissue.
  • CNRG cellularized nerve regeneration graft
  • the cellularized nerve regeneration graft disclosed herein utilizes an optionally multilayered electrospun biodegradable polymer conduit seeded with fibroblasts (FBs) in its exterior surface and Schwann cells (SCs) in its interior luminal space. These Schwann cells may be derived through an induced pluripotent stem cell pathway to minimize graft rejection and facilitate axonal regrowth into a favorable regenerative environment.
  • FBs fibroblasts
  • SCs Schwann cells
  • a cellularized nerve regeneration graft comprising: an electrospun biodegradable polymer conduit having an exterior surface and an interior luminal space; a plurality of fibroblasts seeded to the exterior surface of the conduit; and a system filling the interior luminal space of the conduit.
  • the system comprises a hydrogel matrix or augmented hydrogel matrix and Schwann cells.
  • the cellularized nerve regeneration graft may be a solid structure containing Schwann cells throughout the interior luminal space, with fibroblasts localized on the external surface. The Schwann cells may grow 3 -dimensionally throughout the hydrogel matrix or augmented hydrogel matrix.
  • a patient may be prepared for surgery and the cellularized nerve regeneration graft, containing live cells and growth factors, implanted into the patient by suturing or otherwise adhering the terminal ends of the graft to the distal and proximal ends of the nerve injury.
  • a pre-established cellular structure is developed prior to implantation, such that a pre-established tissue will promote cell survival once implanted and will constitute a “true- to-zri vivo" environment upon implantation.
  • Previous approaches have simply injected Schwann cells into a gel without growing them to a stable culture within the hydrogel prior to implantation. By developing a pre-established tissue-like construct, this will mitigate postimplantation toxic apoptotic byproducts and reinforce the authentic cell-cell interactions present in living tissue.
  • the role of fibroblasts is to establish the basal directionality for Schwann cells and to provide growth and adhesion factors for the Schwann cells, thus mimicking authentic nerve architecture.
  • the electrospun biodegradable polymer conduit may have a diameter of about 1.0 mm to about 5.0 mm, or about 1.5 mm to about 4.0 mm.
  • the conduit may have a length of about 1.0 cm to about 10.0 cm, or about 1.0 cm to about 5.0 cm.
  • the conduit may have a thickness of about 50 pm to about 500 pm, or about 50 pm to about 300 pm.
  • the conduit may be constructed from a tyrosine-derived or tyrosol-derived polymer, which may alternatively be referred to as a tyrosine-polymer, or tyrosol-derived polymer, respectively.
  • the tyrosinederived or tyrosol-derived polymer have non-inflammatory degradation bioproducts.
  • the conduit may be composed of a tyrosine-derived polymer, for example, desaminotyrosyltyrosine ethyl ester (DTE), desaminotyrosyl-tyrosine (DT), or a combination thereof.
  • the conduit may be composed of desaminotyrosyl-tyrosine ethyl ester (DTE), desaminotyrosyl- tyrosine (DT), and polyethylene glycol (PEG).
  • DTE desaminotyrosyl-tyrosine ethyl ester
  • DT desaminotyrosyl- tyrosine
  • PEG polyethylene glycol
  • the molar fraction of free carboxylic acid units and PEG units in the polymer described herein can be adjusted to modify the mechanical properties and degradation rates of NADs made from such polymers. For example, polymers with lower amounts of free carboxylic acid will tend to have longer lifetimes in the body.
  • the resulting polymers can be adapted for use in various applications requiring different device lifetimes.
  • the higher the molar fraction of free carboxylic acid units the shorter the lifetime of the device in the body and more suitable such devices are for applications wherein shorter lifetimes are desirable or required.
  • the conduit may be composed of a tyrosol -derived polymer, for example, U.S. Publication No. 2020/0181321 and WO 2021/055090, which are incorporated by reference herein in their entirety.
  • the conduit may be composed of poly(HTy glutarate), poly(HTy suberate), poly(HTY dodecanedioate), poly(HTy phenylenediacetate), or any combination thereof.
  • the conduit may be composed of poly(HTy glutarate).
  • Formulaa I a biodegradable polymer having repeating units of the structure (Formula I): wherein a and b are independently 0 or an integer between 1 and 6, inclusive; wherein c and d are independently 0 or an integer between 1 and 6, inclusive; wherein each
  • a and b are two and one, respectively.
  • c and d are two and one, respectively, and R 1 is ethyl.
  • R 2 for said polymer is ethylene and k is between about 25 and about 50.
  • the benzyl ester polymers may be converted to the corresponding free carboxylic acid polymers by the palladium catalyzed hydrogenolysis method disclosed in U.S. Pat. No. 6, 120,491.
  • the tert-butyl ester polymers may be converted to the corresponding free carboxylic acid polymers through the selective removal of the tert-butyl groups by the acidolysis method disclosed in U.S. Patent Publication No. 20060034769, also incorporated herein by reference.
  • Polymers may be selected which degrade or resorb within a predetermined time. For this reason, embodiments may include polymers with molar fractions of monomeric repeating units with pendant fee carboxylic acid groups, such as DT, between about 2 and about 20 mol %, and preferably between about 5 and about 20 mol %.
  • Poly(alkylene glycol) segments decrease the surface adhesion of the polymers.
  • the hydrophilic/hydrophobic ratios of the polymers can be changed to adjust the ability of the polymer coatings to modify cellular behavior.
  • Increasing levels of poly(alkylene glycol) inhibit cellular attachment, migration and proliferation.
  • PEG increases the water uptake, and thus increases the rate of degradation of the polymer.
  • polymers are selected in which the amount of poly(alkylene glycol) is limited to between 0.5 and about 10 mol %, and preferably between about 0.5 and about 5 mol %, and more preferably between about 0.5 and about 1 mol %.
  • the poly(alkylene glycol) may have a molecular weight of 1 k to 2 k.
  • the polymer may be selected having intrinsic physical properties appropriate for use in polymer conduits with suitable mechanical properties including elasticity, rigidity, strength and degradation behavior.
  • suitable mechanical properties including elasticity, rigidity, strength and degradation behavior.
  • Such polymers include, if the polymer is amorphous, polymers with a glass transition temperature greater than 37° C. when fully hydrated under physiological conditions and, if the polymer is crystalline, a crystalline melting temperature greater than 37° C. when fully hydrated under physiological conditions.
  • biodegradable and biocompatible polymers can be used to form fibers that provide or reinforce certain desirable properties of the resulting polymer conduits.
  • examples of other polymers that may be used include, but are not limited to, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), polycaprolactone, various poly(amino acid)s and polyanhydrides.
  • Other natural or non-natural fiber materials for example, collagen, cellulose, chitosan, and their derivatives, may alternatively or additionally be utilized to provide or reinforce certain desirable properties of the resulting polymer conduits (see, for example, U.S. Pat. No. 8,216,602).
  • the electrospun biodegradable polymer conduit may be constructed from any polymer disclosed in U.S. Patent Publication No. 2018/0280567, which is incorporated by reference herein in its entirety.
  • the electrospun biodegradable polymer conduit may be constructed from a biocompatible polymer comprising a recurring unit of Formula XVIII: wherein: (a) A is CH 2 or CH 2 CH 2 , B is a bond, Y is selected from the group consisting of (CH 2 ) 2 , (CH 2 ) 3 , CH 2 OCH 2 , (CH 2 ) 4 , CH 2 CH ⁇ CHCH 2 , (CH 2 ) 5 , (CH 2 ) 6 , and (CH 2 ) 10 ; or (b) A is CH 2 CH 2 , B is selected from the group consisting of —O—CO—CH 2 CH 2 , — O—CO—CH 2 CH 2 CH 2 , and —O—CO—CH 2 OCH 2 and bonded to A via oxygen
  • the electrospun biodegradable polymer conduit may be constructed from PEG block polymers of the foregoing polymer.
  • A is C 1-3 alkylene, C 1-3 alkylene–O-CO-C 2-5 alkylene, or C 1-3 alkylene–O- CO-C 1-2 alkylene-O-C 1-2 alkylene. In some embodiments, A is CH 2 or CH 2 CH 2 . In some embodiments, R 1 is H.
  • the amino acid moiety is derived from natural amino acid. In some embodiments, the amino acid moiety is derived from essential amino acid selected from the group consisting of phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine.
  • Y is selected from the group consisting of C 1-5 alkylene, phenylene, and C 1-2 alkylene-O-C 1-2 alkylene.
  • R 2 and R 3 in each occurrence are independently a bromine or iodine; a and b are independently 0, 1 or 2.
  • the biocompatible polymer further includes a recurring unit of the formula II-a: wherein m’ is an integer ranging from 1-3.
  • the biocompatible polymer further includes a recurring unit of the formula II-b: wherein G is C 2-3 -alkylene, n is an integer ranging from 4 to 3000.
  • the biocompatible polymer further includes a recurring unit of the formula II-c: , wherein G is C 2-3 -alkylene, n’ is an integer ranging from 4 to 3000.
  • the biocompatible polymer further includes a copolymer unit selected from the group consisting of poly(ethylene glycol), polycaprolactone-diol, polycaprolactone, poly(trimethylene carbonate), polylactide, polyglycolide, and poly(lactic- co-glycolic acid).
  • A is selected from the group consisting of C 1-3 alkylene, C 1-3 alkylene–O-CO-CH 2 CH 2 , C 1-3 alkylene–O-CO- CH 2 CH 2 CH 2 , and C 1-3 alkylene–O-CO-CH 2 OCH 2 ;
  • B is oxygen.
  • R 1 and R c are H.
  • the electrospun biodegradable polymer conduit may be constructed from PEG block polymers of any of the foregoing biocompatible polymers. [0045] Also incorporated herein by reference in entirety are: U.S. Patent No. 5,099,060, in particular, for its disclosure related to polycarbonate synthesis; U.S.
  • the electrospun biodegradable polymer conduit may be multilayered. It may include one layer, two layers, three layers, or four layers. Each layer may contain aligned (which may also be referred to as oriented) or unaligned (which may also be referred to as non-oriented) biopolymer fibers.
  • the electrospun biodegradable polymer conduit may include two layers, with the inner most layer containing aligned biopolymer fibers, and the outermost layer including unaligned biopolymer fibers.
  • Figures 1A, 1B, 2A and 2B are electron micrograph images of examples of bilayered sheets with oriented layers (e.g., FIGS. 1A and 2A) and unoriented layers (e.g., FIGS.1B and 2B) of biopolymer fibers.
  • the sheets may be rolled and fabricated as electrospun biodegradable polymer conduit of the desired diameter.
  • the fibroblasts may be seeded at a defined concentration, for example, at a density of about 1.0 x 10 5 cells/mL to about 5.0 x 10 5 cells/mL, or about 2.5 x 10 5 cells/mL, onto the exterior surface of the electrospun biodegradable polymer conduit.
  • the fibroblasts adhere to the exterior surface and form a continuous cell layer on the exterior surface at a concentration of about 1.0 x 10 5 cells/cm 2 to about 1.0 x 10 6 cells/cm 2 .
  • the fibroblasts may be epineurial fibroblasts.
  • Fibroblasts and Schwann cells may be obtained through induction of patient cells, such as skin cells, which can be retrieved at the time of patient injury. These skin or other cells, once obtained from the patient, are differentiated into fibroblasts and Schwann cells in vitro and then applied to the electrospun biodegradable polymer conduit as described herein. Use of autologous cells reduces the likelihood of implant rejection because the conduit retains the patient’s genetic material.
  • human Schwann cells may be made from human induced pluripotent stem cells (iPSC) via conversion to human Schwann cell precursor cells (hSCP).
  • iPSC human induced pluripotent stem cells
  • hSCP human Schwann cell precursor cells
  • the system comprising a hydrogel matrix or augmented hydrogel matrix and Schwann cells promotes neurite regrowth from, and myelination of, injured neurons.
  • the hydrogel matrix may be defined as a cross-linked hydrophilic polymer that does not dissolve in water and is capable of absorbing large quantities of water or other biological fluids, which may be made from RADA-16 peptides, collagen, gelatin, alginate, hyaluronic acid, or any other known hydrogel materials.
  • the augmented hydrogel matrix is a hydrogel matrix that is combined via blending, mixing, chemical conjugation, or other known method, with another biochemical factor, such as a growth factor, guidance cue, structural peptide, adhesion factor, other chemical agent, or combination thereof.
  • the augmented hydrogel matrix may be a RADA16 peptide, collagen, gelatin, alginate, or hyaluronic acid hydrogel.
  • the augmented hydrogel matrix may be functionalized with a growth factor to support cell growth, either by physical mixing with the hydrogel matrix or by chemical conjugation with the hydrogel matrix, or any combination thereof.
  • the growth factor may be selected from Neuregulin 1 (NRG1), EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, Erythropoietin (EPO), TPO, BMP, HGF, GDF, Neurotrophins (e.g., GDNF, CNTF, BDNF, NT3), netrins, MSF, SGF, or any combination thereof.
  • the growth factor may be NRG1.
  • the augmented hydrogel matrix may include one or more additives selected from a basal medium known for use in supporting the growth of cells (e.g., Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), an antibiotic (e.g., penicillin, streptomycin or a combination thereof), forskolin, and any combination thereof.
  • a basal medium known for use in supporting the growth of cells
  • DMEM Dulbecco’s Modified Eagle Medium
  • FBS fetal bovine serum
  • an antibiotic e.g., penicillin, streptomycin or a combination thereof
  • forskolin e.g., forskolin, and any combination thereof.
  • the Schwann cells may be substantially evenly distributed throughout the hydrogel matrix or augmented hydrogel matrix within the interior luminal space of the conduit.
  • the Schwann cells may be distributed throughout the hydrogel matrix or augmented hydrogel matrix at a concentration of about 5 million cells/mL to about 80 million cells/mL, about 15 million cells/mL to about 75 million cells/mL, or about 20 million cells/mL to about 70 million cells/mL.
  • the Schwann cells may be encapsulated by the hydrogel matrix or augmented hydrogel matrix within the interior luminal space of the conduit.
  • FIGS. 14A-C are cross- sectional confocal microscopy images demonstrating rat Schwann cells proliferating within collagen hydrogel matrix. In these figures, tyrosine-derived polymer fibers are shown dispersed throughout the matrix, maintaining longitudinal tracts in the hydrogel matrix. In FIG.
  • FIGS. 14A depth coding of fibers and Schwann cells are distributed in three dimensions as indicated by red-blue scale corresponding to depth within the hydrogel matrix.
  • FIGS.14B and C tyrosine-derived fibers shown as the large uniform circular structures; Schwann cell nuclei are shown as smaller points, stained with DAPI, interspersed within hydrogel matrix.
  • FIGS. 3A-E depict hydrogel encapsulated Schwann cells in an electrospun biodegradable polymer conduit.
  • FIG.3A a 96-well optical plate is used to hold and culture upright electrospun biodegradable polymer conduits filled with Schwann cells encapsulated in hydrogel enabling live imaging;
  • FIG.3A a 96-well optical plate is used to hold and culture upright electrospun biodegradable polymer conduits filled with Schwann cells encapsulated in hydrogel enabling live imaging;
  • FIG.3A a 96-well optical plate is used to hold and culture upright electrospun biodegradable poly
  • FIG.3B shows GFP (Green Fluorescent Protein)+ rat Schwann cells (SC) and human neural progenitor cells embedded in 3-dimensional RADA16 peptide hydrogel within the electrospun biodegradable polymer conduit.
  • FIG.3C is a close up panel B showing GFP+ rat SC near tube edge and DIC to show conduit edge and outer well space.
  • FIG 3D is a close up of panel B showing only GFP+ SC.
  • FIG.3E is an example of a 3- dimensional reconstruction of a 32 ⁇ M Z-stack showing Rat SC (GFP+) embedded in hydrogel enclosed by the electrospun biodegradable polymer conduit.
  • the system may also contain fibroblasts, which may be substantially evenly distributed throughout the hydrogel matrix or augmented hydrogel matrix within the interior luminal space of the conduit.
  • the fibroblasts may be present at a lower concentration than the Schwann cells, for example at about a 2:10 to about 1:20, or about 1:10 ratio of fibroblasts to Schwann cells.
  • the fibroblasts facilitate Schwann cell function within the cellularized nerve regeneration graft, particularly at the level of the endoneurium, the innermost connective tissue layer found within nerve fascicles that surround myelinated axons.
  • the system may contain other supporting cells, optionally in addition to fibroblasts, such as, but not limited to, endothelial cells, or other cells to support the graft cellular architecture.
  • the inner luminal space of the polymer conduit may be filled with hydrogel matrix or augmented hydrogel matrix with rat SC growing throughout.
  • GFP+ fibroblasts may be only seeded on the exterior surface of the polymer conduit, therefore no GFP signal should be present within the inner luminal space of the polymer conduit.
  • fibers or hollow tubes optionally having a diameter of about 5 ⁇ m to about 50 ⁇ m, or about 20 ⁇ m, may be added to reproduce an endoneurial sheath-like substructure.
  • the fibers or hollow tubes may be made from collagen, or other suitable fibers, including fast-degrading polymeric fibers, or water- soluble sacrificial fibers made of materials, such as sucrose or other suitable saccharide, or made by creating channels with metallic or polymeric wires. Creation of an endoneurial sheath- like substructure may augment cell-cell signaling during graft development and after implantation. [0057] Another embodiment is a method of making a cellularized nerve regeneration graft.
  • the nerve call graft comprising: an electrospun biodegradable polymer conduit having an exterior surface and an interior luminal space; a plurality of fibroblasts seeded to the exterior surface of the conduit; and a system filling the interior luminal space of the conduit, the system comprising a hydrogel matrix or augmented hydrogel matrix and Schwann cells.
  • the method may include: electrospinning a polymer formulation, e.g., a tyrosine-derived or tyrosol-derived polymer, to make the electrospun biodegradable polymer conduit; culturing fibroblasts; seeding the fibroblasts on the exterior surface of the electrospun biodegradable polymer conduit; generating human Schwann cells; embedding the human Schwann cells in the hydrogel matrix or augmented hydrogel matrix to make the system; seeding the system in the interior luminal space of the electrospun biodegradable polymer conduit to make a seeded conduit; and incubating the seeded conduit for about 1 to about 6 weeks, or about 2 to about 4 weeks in the system to make the cellularized nerve regeneration graft.
  • a polymer formulation e.g., a tyrosine-derived or tyrosol-derived polymer
  • the fibroblasts may be cultured by any method known in the art.
  • the fibroblasts may be epineurial fibroblasts.
  • the exterior surface of an electrospun tyrosine-derived polymer conduit may be seeded with epineurial fibroblasts.
  • the exterior surface of an electrospun tyrosol-derived polymer conduit may be seeded with epineurial fibroblasts.
  • Generating human Schwann cells may be accomplished by any means known in the art.
  • the generated human Schwann cells may be embedded in the hydrogel matrix or augmented hydrogel matrix, which is optionally functionalized with one or more growth factors to support axonal regrowth, Schwann cell proliferation, and myelination of axonal projections.
  • a hydrogel matrix or augmented hydrogel matrix at a defined concentration may be mixed with Schwann cells (SCs) at a defined concentration (e.g., about 1 million cells/mL to about 20 million cells/mL, about 2 million cells/mL to about 15 million cells/mL, about 5 million cells/mL to about 10 million cells/mL or about 10 million cells/mL) to create a system.
  • SCs Schwann cells
  • the system may be injected into the interior luminal space of the electrospun biodegradable polymer conduit, thus filling the entire interior luminal space.
  • the filled electrospun biodegradable polymer conduit may be incubated in the system for about 2 to about 4 weeks, or about 3 weeks, to allow both epineurial fibroblasts and Schwann cells to proliferate within the conduit.
  • a media solution may be added and routinely replaced during the incubation.
  • the media solution may be Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), an antibiotic (e.g., penicillin, streptomycin or a combination thereof), and any combination thereof.
  • the media solution may be DMEM/10% FBS (Fetal Bovine Serum)/1%Penicllin/Streptomycin, optionally supplemented with forskolin and/or a growth factor at defined concentrations.
  • Forskolin may be added at a concentration of about 1 ⁇ M to about 5 ⁇ M, or about 2 ⁇ M.
  • the growth factor may be neuregulin-1, and may be added at a concentration of about 5 ng/ml to about 15 ng/ml, or about 10 ng/ml.
  • the media solution may be 1.2 mL DMEM/10%FBS/Penicillin/streptomycin.
  • the fibroblasts and Schwann cells may be grown to confluence in the media solution such that, at the end of this period, the fibroblasts constitute an outer contiguous cell layer at a defined concentration of about 1.0 x10 5 cells/cm 2 to about 1.0 x10 6 cells/cm 2 on the exterior surface of the conduit while, in the interior luminal space, there may be a confluent, 3D, substantially even distribution of Schwann cells throughout the hydrogel matrix or augmented hydrogel matrix such that the Schwann cells occupy the entire luminal space of the conduit at a defined concentration of about 5 million cells/mL to about 80 million cells/mL, about 15 million cells/mL to about 75 million cells/mL, or about 20 million cells/mL to about 70 million cells/mL.
  • a microdevice may be made by 3-D printing and assembled.
  • the 3-D printed microdevice may include a number of parts including one or more rods and one or more gears, which are constructed as separate pieces but which may be assembled, or reversibly or irreversibly interlocked, to create a microdevice for seeding the fibroblasts.
  • a 3-D printed microdevice may also be used to seed the system in the interior luminal space of the electrospun biodegradable polymer conduit.
  • the 3-D printed microdevice may be the same microdevice used for seeding the fibroblasts.
  • a microdevice may be assembled from a rod (Figure 4A), a hollow gear (Figure 4B), a solid gear (Figure 4C), and capped hollow tube (Figure 4E).
  • the rod may be inserted through hollow gear.
  • the rod may have a diameter of about 1.0 mm to about 4.0 mm, depending on the application, with an overall length of about 5.0 mm to about 5.0 cm, or greater.
  • Each of the hollow gear and solid gear may have a diameter of about 8.0 mm to about 2.0 cm with a thickness of about 1.0 mm to about 2.0 mm.
  • Each of the components of the 3-D printed microdevice may be constructed from polylactic acid (PLA), polycaprolactone (PCL), or any other material known for use in the art.
  • the electrospun biodegradable polymer conduit may be then placed over (or around) the rod and capped with the solid gear, resulting in an assembly, as shown in Figure 4D, with the electrospun biodegradable polymer conduit shown in transparent shading.
  • the assembly, as shown in Figure 3D may then placed into a well plate (shown below in Figure 8E) and seeded with fibroblast suspension at a determined concentration, e.g., about 1.0 x 10 5 cells/mL to about 5.0 x 10 5 cells/mL, or about 2.5 x 10 5 cells/mL.
  • the assembly After being submerged in the culture media within the well plate, the assembly may be permitted to rest for about 2 to about 4 minutes to allow the fibroblasts to attach to the scaffold and then the assembly may be rotated about its longitudinal axis by 90 degrees while still inside of the well. This process may be repeated three times until four arc lengths of the conduit is seeded with fibroblast suspension.
  • the assembly may be then submerged in a media solution by pipetting fresh media into the bottom of the well until the assembly was completely covered by media.
  • the assembly After seeding the exterior surface with fibroblasts, which may take a period of about 1 hour to about 24 hours, the assembly may be removed from the well and placed upright, with the hollow gear resting on a capped hollow tube in an adjacent well.
  • the solid gear may be removed from the assembly and, optionally, discarded or reused after sterilization. Pressure may be applied from above to the rod which forces the rod through the hollow gear and into the capped hollow tube, resulting in the interior of the biopolymer tube (or conduit) being exposed, as shown in FIG.4F.
  • the device shown in FIG.4F may then be placed into any known culture tube, such as a polystyrene culture tube, and the system (e.g., the mixture of the hydrogel matrix or augmented hydrogel matrix and Schwann cells) may be inserted into the interior luminal space of the electrospun biodegradable polymer conduit that has been seeded with fibroblasts on the exterior surface.
  • a media solution may be added to the culture tube to submerge the device.
  • the culture tube may be capped and incubated, for example at about 33-40 degrees C. and about 3%-7% CO 2 , or about 37 degrees C. and about 5% CO2 for about 15 to about 30 minutes, or about 20 minutes.
  • After a first period of incubation at least a portion of the media solution may be replaced, and this process may be repeated 2-4 times every about 15 to about 30 minutes, or replaced about every 20 minutes. Incubation may then be permitted for about 3 weeks, with at least a portion of the media solution in the culture tube being replaced about every 2-3 days.
  • the filled and seeded electrospun biopolymer conduit is a solid graft-like structure containing Schwann cells throughout the lumen, with fibroblasts localized externally.
  • the fibroblasts may form the outer epineurial sheath, a layer of connective tissue that encloses fascicles of peripheral nerves.
  • the fibroblasts serve the purpose of directing the apical/basal orientation of Schwann cells while also secreting growth factors to support Schwann cell survival in vitro and in vivo.
  • a method for repairing an injured peripheral nerve comprising implanting a cellularized nerve regeneration graft into a patient by adhering the cellularized nerve regeneration graft to a distal end and a proximal end of the injured nerve injury.
  • the cellularized nerve regeneration graft comprises: an electrospun biodegradable polymer conduit having an exterior surface and an interior luminal space; a plurality of fibroblasts seeded to the exterior surface of the conduit; and a system filling the interior luminal space of the conduit, the system comprising a hydrogel matrix or augmented hydrogel matrix and Schwann cells.
  • the Schwann cells which occupy the interior of the cellularized nerve regeneration graft, serve the purpose of supporting in-growing axons by secreting signaling molecules and growth factors, including ciliary neurotrophic factor (CNTF), to guide axon growth through the graft and to myelinate regenerating axons once the graft is implanted into the patient.
  • CNTF ciliary neurotrophic factor
  • resorbable fibers which may be about 10 ⁇ m to about 50 ⁇ m in diameter
  • the resorbable fibers may be made of: poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), a tyrosine-derived or tyrosol- derived polymer with a high concentration of poly(ethylene glycol) (PEG), or any combination thereof.
  • PLGA poly(lactic-co-glycolic acid)
  • PGA polyglycolic acid
  • PEG poly(ethylene glycol)
  • One or more coating materials may be applied to the surface of the resorbable fibers.
  • the coating material may be selected from: poly-d-lysine or other charged molecules; macromolecular coatings such as collagen hydrogel, hyaluronic acid or others which may mimic the constituents of the extracellular matrix; and any combination thereof.
  • Cells in suspension may be applied to the resorbable fibers and may be assisted by the action of capillary action to coat the fibers evenly.
  • Figure 13 is an example of 10X magnification imaging of rat Schwann cells growing on E1001k fibers of approximately 50 ⁇ m.
  • the resorbable fibers cultured with the Schwann cells may be loaded into a biodegradable polymer conduit in a longitudinal arrangement (alternatively, the fibers may be present within the biodegradable polymer conduit at the time of cell seeding), with a spacing between the fibers of about 20 ⁇ m to about 100 ⁇ m.
  • the remaining luminal space may be filled with the hydrogel matrix or augmented hydrogel matrix, such that the interior consists of parallel-oriented fibers surrounded by Schwann cells, or with Schwann cells directly adhered to the resorbable fibers, and the remainder of the luminal space occupied by the hydrogel matrix or augmented hydrogel matrix, or a hydrogel matrix or augmented hydrogel matrix mixed with cells, such as but not limited to, Schwann cells, fibroblasts and/or other cells.
  • the hydrogel matrix or augmented hydrogel matrix such that the interior consists of parallel-oriented fibers surrounded by Schwann cells, or with Schwann cells directly adhered to the resorbable fibers, and the remainder of the luminal space occupied by the hydrogel matrix or augmented hydrogel matrix, or a hydrogel matrix or augmented hydrogel matrix mixed with cells, such as but not limited to, Schwann cells, fibroblasts and/or other cells.
  • the remaining luminal space may be filled with the hydrogel matrix or augmented hydrogel matrix and cells mixture, optionally having a ratio of approximately 3:2 (hydrogel:cells) in volume.
  • the resorbable fibers dissolve, leaving Schwann cells within channels running longitudinally within the hydrogel matrix or augmented hydrogel matrix.
  • the resorbable fibers may also constitute such a composition that an inner, quickly dissolving material, such as, but not limited to, sucrose, poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), or a combination thereof, is coated with resorbable material, such as, but not limited to, PLGA, PGA, tyrosine-, or tyrosol-derived polymers with large amounts of PEG, or a combination thereof which dissolves over a longer time period such that the arrangement results in a hollow channel with adherent/surrounding cells.
  • PVA poly(vinyl alcohol)
  • PEG poly(ethylene glycol)
  • resorbable material such as, but not limited to, PLGA, PGA, tyrosine-, or tyrosol-derived polymers with large amounts of PEG, or a combination thereof which dissolves over a longer time period such that the arrangement results in a hollow channel with adherent/surrounding cells.
  • These channels serve as the
  • the hollow channels may contain Schwann cells within the longitudinal space. This method may be employed to create channels without Schwann cells, such that nutrients and culture media can traverse the length of the conduit and diffuse throughout the hydrogel matrix or augmented hydrogel matrix.
  • FIG.5 is an example of the method of forming a plurality of channels reinforced with Schwann cells within a hydrogel matrix or augmented hydrogel matrix.
  • a biodegradable polymer conduit 1 also referred to as a scaffold tube, is shown filled with resorbable fibers 2.
  • a suspension 3 with Schwann cells is seeded onto the resorbable fibers and the cells allowed to adhere to the fibers to make SC coated fibers 4.
  • the remaining luminal space within the biodegradable polymer conduit is filled with a suspension of hydrogel or augmented hydrogel matrix, optionally including Schwann cells 5.
  • a suspension of hydrogel or augmented hydrogel matrix optionally including Schwann cells 5.
  • the resorbable fibers degrade leaving channels 7 reinforced by Schwann cells, with the channels running longitudinally and for in-growing axons during nerve regeneration.
  • the biodegradable polymer conduit 1 includes an outer unoriented electrospun layer 10 and an inner oriented electrospun layer 11.
  • EXAMPLE 1 Preparation of Tyrosine-derived polymer electrospun scaffold: Scaffolds were prepared from the random block copolymer poly(DTE-co-10% DT-co-1% PEG carbonate) composed of desaminotyrosyl-tyrosine ethyl ester (DTE), desaminotyrosyl-tyrosine (DT), and polyethylene glycol (PEG), that will be referred to as E1001(1k), where 10 and 01 are the mole percent of DT and PEG, respectively, and 1k is the molecular weight of PEG (1000 Da) 5,6.
  • DTE desaminotyrosyl-tyrosine ethyl ester
  • PEG polyethylene glycol
  • the three components of the polymer serve different purposes.
  • the main chain of DTE segments aids in polymer processing, has the required mechanical properties, and provides chemical stability during processing and use.
  • Increasing the fraction of DT units increases the degradation rate from days at 25 mol% DT to hours at 50 mol% DT.10 mol% DT used here provides a degradation rate of approximately 1 year.
  • PEG was incorporated to increase water content and allow for degradation.
  • PEG(1k) remains biocompatible after degradation and, unlike PEG(2k), does not crystallize in the scaffold.
  • the polymer was dissolved in hexafluoropropylene to prepare a 16% solution.
  • the electrospinning apparatus consisted of a syringe pump (kd Scientific, Model 780100, Holliston, MA), high voltage DC power supply (Gamma High Voltage Research, Model ES30P/5W/DAM, Ormand Beach, FL) fitted with an 18 G needle, and a rotating mandrel. The syringe was placed 10 cm away from the mandrel.
  • Rat Schwann cell culture Primary rat Schwann cells (SC) were cultured on Matrigel (Corning) coated plates in DMEM (Dulbecco's Modified Eagle’s Medium) supplemented with 10% FBS, 1% Penicillin/Streptomycin, 2 ⁇ M Forskolin, and 10 ng/ml Neuregulin-1 (NRG1). Rat SC were routinely passaged using Accutase (StemCell Technologies).
  • iPSC human induced pluripotent stem cells
  • human fibroblasts To generate human induced Schwann cells, human iPSC were passaged with Accutase (StemCell Technologies) and plated on growth-factor-reduced Matrigel (Corning) plates in induction medium containing 1:1 DMEM/F12 (Hyclone) and neurobasal medium (Gibco) supplemented with 1X B27 (Gibco), 3 ⁇ M CHIR99021 (StemCell Technologies), 20 ⁇ M SB431542 (StemCell Technologies), and 50 ng/ml Neuregulin-1 (Peprotech) for 18 days with media changes every other day.
  • induction medium containing 1:1 DMEM/F12 (Hyclone) and neurobasal medium (Gibco) supplemented with 1X B27 (Gibco), 3 ⁇ M CHIR99021 (StemCell Technologies), 20 ⁇ M SB431542 (StemCell Technologies), and 50 ng
  • the media was changed to 1:1 DMEM/F12 and neurobasal medium supplemented with 1X B27, 200 ng/ml Neuregulin- 1, 4 ⁇ M Forskolin (Sigma), 10 ng/ml PDFG-BB (Peprotech), and 100 nm all-trans retinoic acid (Sigma) for 3 days. After 3 days, the same medium was given minus the all-trans retinoic acid and forskolin and cultured for 3 more days. The induced Schwann cells were then maintained in 1:1 DMEM/F12 and neurobasal medium supplemented with 1X B27 and 200 ng/ml Neuregulin-1 until ready for experiments and fed every 3-4 days.
  • Encapsulation of Schwann cells in hydrogel To encapsulate Schwann cells in RADA16 peptide hydrogel, either rat Schwann cells or human-induced Schwann cells were dissociated with Accutase, resuspended in 10% sucrose water, and counted with a hemocytometer to ensure proper loading density of about 1 million cells/ml to about 20 million cells/ml. RADA16 peptide hydrogel was mixed 1:1 with 20% sucrose water.
  • the cell suspension and hydrogel mixture were then combined 1:1, briefly mixed, and loaded in an upright electrospun biodegradable polymer conduit.
  • the final concentration of the hydrogel is 0.25%.
  • media composing DMEM, 10% FBS, 1% Penicillin/Streptomycin, 2 ⁇ M Forskolin, and 10 ng/ml Neuregulin-1 was added to the well to initiate curing of the hydrogel.
  • Media was replaced after the first 20 minutes to reduce acute acidity caused by the hydrogel.
  • Electrospun biodegradable polymer conduits containing the hydrogel encapsulated Schwann cells were then cultured in an incubator at 37 ⁇ C 5% CO 2 .
  • a biopolymer conduit (tube) using electrospun tyrosine-derived polymer as the substrate; 2) culture epineurial fibroblasts (FBs) and seed them on the outer surface of the conduit using the microdevice that was developed (see FIGS.7C-E and 4A-F); 3) generate Schwann Cells (SCs), which are the myelinating cells for peripheral nerves, and coat them onto resorbable fibers and/or mix them with functionalized hydrogel (with growth factors); and 4) seed the mixture of hydrogel/SCs in the inner space of the biodegradable polymer conduits using the microdevice shown in Figure 7C-7E.
  • SCs Schwann Cells
  • the cellularized nerve regeneration graft will be cultured in culture dishes for about 1-6 weeks before grafting into the lesion site to help facilitate axonal regeneration, myelination, and function.
  • Manufacturing of biopolymer and assemblies for Cellularized Nerve Regeneration Graft [0083] Flat sheets were made using a large diameter (5 cm) mandrel that was laterally oscillated to obtain 13 x 21 cm mats. The speed of the mandrel was controlled by a DC power supply (Model 1627A, BK Precision, Yorba Linda, CA). The linear speed was set at 30 meters per minute (mpm) for unaligned layers and 650 mpm for aligned layers.
  • Multi-layered scaffolds were prepared in three steps: 16% polymer solution was spun into an unaligned layer at 2 mL/h for 30 minutes, followed by an additional unaligned layer with 10% solution at 1 mL/h for 30 minutes, and finally an aligned layer with 10% solution at 1 mL/h for 30 minutes. [0085] Initially, these multilayer scaffolds were electrospun as flat sheets, dried slowly at RT (Room Temperature), and refrigerated at 4 degrees C until needed. Shortly prior to culture experiments, the polymer was cut into 8-mm circular sections (as shown in FIG.7F), as needed, using an 8-mm diameter steel hollow punch.
  • Cut scaffolds were secured into the snap-fit fixture shown in FIG.7C-7E after sterilizing all components under UV light for 30 minutes.
  • hollow biopolymer conduits were then prepared using smaller diameter (1.5, 2, and 4 mm) mandrels.
  • the mandrels were coated with PEG gel to facilitate the release of the conduit after electrospinning.
  • These mandrels were mounted onto a chuck (IKA, model R20DS1) and spun at approximately 200 rpm for 10 min to 2 h to obtain tubes of different wall thicknesses and tube diameters.
  • the mandrel was removed from the chuck, wetted slightly with deionized water, and the polymer conduit was carefully removed by sliding it off the mandrel. Conduits were allowed to dry slowly and then refrigerated at 4 degrees C, until needed, to prevent decomposition. For culture experiments, shortly prior to cell seeding the conduits were cut into 5-mm length sections using stainless steel surgical scissors and then sterilized under UV light for 30 minutes. Scaffold thickness was measured using a micrometer. Fiber morphology was assessed using a scanning electron microscope (SEM) (Phenom ProX, Nanoscience Instruments, Phoenix, AZ).
  • SEM scanning electron microscope
  • FIG.7A SEM image of unaligned E1001(k) biopolymer fibers forming flat electrospun sheet, at 500x magnification is shown in FIG.7A.
  • FIG.7B SEM image of the same electrospun sheet in FIG. 7A but at 5000x magnification is shown in FIG.7B.
  • FIG.7C shows a bottom half of 3D-printed snap fixture for holding flat biopolymer sheet.
  • An 8-mm diameter piece of electrospun E1001(k) scaffold may be placed into the round inset shown within the device.
  • FIG 7D shows a top half of 3D-printed snap fixture.
  • FIG.7E This fully assembled configuration is shown in FIG.7E.
  • cell suspension can be pipetted into the wells created on either side of the snap fixture. This allows cells to adhere to only one side of the scaffold fibers while preventing cell migration to the other side.
  • the device is sized with an outer width (from tab to tab) of about 12 mm such that the entire device fits into, and can be turned inside, a well of a standard 24-well culture dish. Holes through the side walls allow for culture media to flow through the device. Indentations in the tabs allow for handling of the device using forceps.
  • FIG. 7F is a sample circular 8-mm DTE biopolymer sheet.
  • a device was assembled such that: a rod (Figure 4A) was inserted through hollow gear (Figure 4B). The electrospun biopolymer tube was then placed over the rod and capped with solid gear ( Figure 4C), resulting in an assembly, as shown in Figure 4D, with the electrospun biopolymer tube here shown in transparent shading.
  • the assembly was then placed longitudinally into a 24-well plate (shown below in Figure 8E) and seeded with 40 uL of fibroblast suspension at a density of 2.5 x 10 5 cells/mL by pipetting the volume across the length of the exterior of the exposed electrospun biopolymer tube.
  • the cells were allowed to attach to the scaffold for 2 minutes and then the device was rotated about its longitudinal axis by 90 degrees by applying force to the tab of one of the gears with a pair of forceps while the device was still inside of the well. This process was repeated three times until four arc lengths of the tube were seeded with fibroblast suspension.
  • the assembly was then submerged in 1.2 mL DMEM/10%FBS/1%P/S by pipetting fresh media into the bottom of the well until the assembly was completely covered by media. After fibroblast cell attachment overnight, the assembly was removed intact from the well with a pair of forceps and placed upright, with the hollow gear (shown in Figure 4B) resting on a capped hollow tube (shown in Figure 4E) in an adjacent well of the 24-well plate. The solid gear was then removed from the assembly with a pair of forceps and discarded. Using the forceps, pressure was applied directly from above to the rod which forced the rod through hollow gear and into the capped hollow tube, resulting in the interior of the electrospun biopolymer tube being exposed, as shown in Figure 4F.
  • the entire device was then placed carefully into a polystyrene culture tube and a hydrogel-Schwann cell mixture was pipetted into the interior lumen of the electrospun biopolymer tube.
  • a hydrogel-Schwann cell mixture was pipetted into the interior lumen of the electrospun biopolymer tube.
  • 1.5 mL of culture media was added to the polystyrene tube to submerge the entire device.
  • the tube was then capped and incubated at 37 degrees C and 5% CO2 for 20 minutes. After 20 minutes 1.0 mL of the media was replaced, and this process was repeated after 40 minutes.
  • the culture media in the tube was replaced similarly every 2-3 days over the course of three weeks.
  • FIG.8A is an SEM image of exterior surface of biopolymer conduit, 500x magnification.
  • FIG 8B is an SEM image of the exterior surface of the same biopolymer conduit as in FIG.8A, but at 5000x.
  • FIG 8C is a 10-mm length of tyrosine-derived polymer conduit.
  • FIG.8D is a photograph of a gear and rod assembly as shown in Figure 4D (here shown without biopolymer conduit).
  • FIG.8E is a photograph of a gear and rod assembly with biopolymer conduit seeded with fibroblasts submerged in culture media within a standard 24-well plate. A volume of 40 ⁇ L of fibroblast suspension was pipetted across the length of the exposed polymer conduit at a density of 2.5 x 10 5 cells/mL.
  • iPSCs are plated on growth-factor-reduced Matrigel plates in a cocktail of 20 ⁇ M SB431542, and 50 ng/ml Neuregulin-1 for 18 days.
  • the cocktail was changed to 200 ng/ml Neuregulin-1, 4 ⁇ M Forskolin, 10 ng/ml PDFG-BB, and 100 nm all-trans retinoic acid for 3 days.
  • the same medium was given minus the all-trans retinoic acid and forskolin and cultured for 3 more days.
  • the induced Schwann cells were then maintained in 50 ng/ml Neuregulin-1 until ready for experiments.
  • Figure 9B shows brightfield images of cells at various stages of the differentiation process.
  • FIGS 11A-C show a diagrammatic representation of Schwann cells and fibroblasts cultured onto opposite sides of an electrospun biodegradable polymer scaffold.
  • one cell type here Schwann cells
  • FIG 11A one cell type (here Schwann cells) is first cultured onto the top layer of the fibers.
  • the scaffold is then inverted, and another cell type (here fibroblasts) is cultured onto the bottom layer of the scaffold.
  • the intervening scaffold localizes the cells to their respective sides while preventing migration of either cell to the opposing side.
  • human fibroblasts have grown onto the unaligned fibers of the biopolymer scaffold; note the multidirectional projects of the actin cytoskeleton as shown by Texas Red phalloidin stain.
  • rat Schwann cells blue nuclei and red actin cytoskeleton are shown growing in a linear alignment along the biopolymer fibers shown in green.
  • epineural FBs are cultured on the outer surface of the biopolymer tube and SCs are cultured in functionalized hydrogel and the hydrogel seeded inside the tube to form a solid structure with SCs cultured in the 3D matrix inside the tube, the biopolymer walls and then the epineural FBs on the outside wall of the biopolymer.
  • SCs are cultured in functionalized hydrogel and the hydrogel seeded inside the tube to form a solid structure with SCs cultured in the 3D matrix inside the tube, the biopolymer walls and then the epineural FBs on the outside wall of the biopolymer.
  • BDNF Brain-Derived Neurotrophic Factor
  • NT3 Neurotrophin-3
  • GDNF Glial-Derived Neurotrophic Factor
  • FIG. 12 depicts a general scheme for implantation of CNRG into a murine peripheral nerve injury model. It shows in vitro modeling of axon growth through the CNRG. Axonal projections are expected to grow through the hydrogel and become myelinated by Schwann cells.
  • Fibroblasts and non-myelinating Schwann cells will secrete ECM (extracellular matrix) components and growth factors to distinct layers of the CNRG. Once cells have grown to confluence within the CNRG, it will be surgically inserted into a murine peripheral nerve injury model. Recovery of motor and sensory capabilities will be assessed in CNRG-treated, autograft-treated and sham animals. [00103] In conclusion, the goal of the study was to construct the CNRG that is suitable for reconstructing and repairing peripheral nerve damage.

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Abstract

L'invention divulgue une greffe de régénération nerveuse cellularisée qui comprend un conduit polymère biodégradable électrofilé présentant une surface extérieure et un espace luminal intérieur, une pluralité de fibroblastes ensemencés sur la surface extérieure du conduit, et un système remplissant l'espace luminal intérieur du conduit. Le système peut comprendre une matrice d'hydrogel ou une matrice d'hydrogel augmentée et des cellules de Schwann. La greffe de régénération nerveuse cellularisée peut être utilisée dans la réparation de lésions nerveuses périphériques. L'invention concerne en outre des méthodes de production de la greffe de régénération nerveuse cellularisée.
EP22877515.1A 2021-09-28 2022-09-28 Greffe de régénération nerveuse cellularisée et ses méthodes de production Pending EP4408493A4 (fr)

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